Process for purifying pravastatin sodium from a fermentation broth

ABSTRACT

A process for purifying pravastatin sodium comprising the steps of:  
     a. clarifying a fermentation broth containing pravastatin sodium to obtain a clear solution; and adjusting the clear solution to be basic having pH value ranging from pH 10˜13;  
     b. adsorbing the pravastatin sodium with non-ionic resin; eluting the pravastatin sodium by water-soluble organic solvent; and forming a concentrate of pravastatin sodium;  
     c. treating the concentrate with water-soluble anti-solvent or inorganic salt to form a precipitate of pravastatin sodium; and  
     d. recrystallizing the precipitate for making pravastatin sodium crystal with high purity and high yield.

BACKGROUND OF THE INVENTION

[0001] U.S. Pat. No. 4,346,227 to Terahara et al. disclosed apravastatin which can be obtained by fermentation of compactin using avariety of microorganisms. After fermentation, pravastatin was separatedfrom the fermentation broth by acidifying the broth to a pH of 3 andextracting pravastatin and other non-hydrophilic organics with ethylacetate, followed by washing with brine. The pravastatin free acid waslactonized by addition of a catalytic amount of trifluoroacetic acid,then neutralized with dilute sodium bicarbonate, dried over sodiumsulfate and evaporated to dryness. The residue was purified bypreparative reverse-phase high performance liquid chromatography.However, such a reverse-phase HPLC is not an economical method ofpurification for large-scale preparation of a chemical compound asevaluated by those skilled in the art.

[0002] Meanwhile, the pravastatin is unstable in the acidic aqueoussolution and will be easily decomposed into 3-Hydroxy-iso-compactin orpravastatin lactone or other impurities. During the extraction for thepurification of pravastatin sodium, emulsion phenomena will also occurto deteriorate the product purity and quality. Therefore, the process ofthe prior art is not suitable for commercial mass production.

[0003] The present inventor has found the drawbacks of the prior art andinvented the present process for stably purifying pravastatin sodiumfrom a fermentation broth.

SUMMARY OF THE INVENTION

[0004] The object of the present invention is to provide a process forstably purifying pravastatin sodium comprising the steps of:

[0005] a. clarifying a fermentation broth containing pravastatin sodiumby centrifugation or filtration to obtain a supernatant or filtratecontaining the pravastatin sodium; and adjusting the supernatant orfiltrate to be basic having pH value ranging from pH 10˜13;

[0006] b. adsorbing the pravastatin sodium with non-ionic resin which isthen washed with water; eluting the pravastatin sodium as adsorbed onthe resin by water-soluble organic solvent to obtain pravastatin sodiumfraction; and concentrating the fraction to be pravastatin sodiumconcentrate;

[0007] c. treating the concentrate with water-soluble anti-solvent orinorganic salt to form a precipitate of pravastatin sodium; and

[0008] d. recrystallizing the precipitate for making pravastatin sodiumcrystal with high purity and high yield.

DETAILED DESCRIPTION

[0009] For isolation and adsorption of pravastatin sodium from afermentation broth by non-ionic resin, it is found that the adsorptionis very poor when the fermentation broth is placed in neutral condition.

[0010] The adsorption efficiency may be increased when the fermentationbroth is adjusted to be acidic or basic. However, the pravastatin isunstable in acidic aqueous solution as aforementioned.

[0011] Surprisingly, the adsorption efficiency of pravastatin sodium onnon-ionic resin will be greatly increased when the fermentation broth isadjusted to be basic ranging from pH 10˜13, preferably ranging from pH11˜12 in accordance with the present invention.

[0012] For clarifying the fermentation broth, centrifugation orfiltration is conducted to obtain a clear solution of supernatant orfiltrate which is adjusted to be basic of pH 10˜13.

[0013] The non-ionic resin is provided to adsorb the pravastatin sodium,which is then washed by water. Water-soluble organic solvent is providedto elute the pravastatin sodium on the resin to obtain pravastatinsodium fraction.

[0014] The non-ionic resin may be selected from Amberlite ®XA-D series,Diaion ®HP or SP series, but not limited in the present invention.

[0015] The adsorption of pravastatin sodium with non-ionic resin underbasic condition in accordance with the present invention will overcomethe drawbacks of instability of pravastatin when existing in acidiccondition and the emulsion phenomena which occurs in conventionalextraction process.

[0016] The pravastatin sodium as obtained through resin adsorption,water-wash and desorption steps of the present invention will render ahigh HPLC purity more than 93%, and a high yield more than 98%.

[0017] The HPLC detection parameters are shown as follows:

[0018] Mobile phase:Methanol:water:acetic acid:triethylamine=500:500:1:1

[0019] Column: Lichroshper RP-18e5.0μ, ¢ 4.0 mn×25 cm (Merck & Co.)

[0020] Column temperature: 40° C.

[0021] Flow rate: 1 ml/min

[0022] Detection wave length: UV: 238 nm

[0023] Under the basic condition (pH 11˜12), the adsorption ordesorption with non-ionic resin is performed in the present invention toobtain high purity and high yield of pravastatin sodium. Parallelly, theimpurity of 6α-hydroxy-compactin sodium (epimer of pravastatin sodium)existing in the broth may be minimized, thereby increasing the overallpurity and yield of the pravastatin sodium in accordance with thepresent invention. Such an epimer, having physical and chemicalproperties very similar to that of pravastatin, will not be easilyremoved by conventional crystallization, chromatography or even highperformance liquid chromatography (HPLC).

[0024] However, due to the disclosure of the critical condition byadjusting the fermentation broth to be basic (pH 10˜13, preferably pH11˜12) as disclosed in the present invention, the “obstacle” (theimpurity epimer) hindering the mass production for purifying pravastatinsodium will be removed.

[0025] After resin adsorption, precipitation and crystallization may beapplied to further purify the pravastatin sodium.

[0026] The eluant (with pravastatin sodium fraction) after the elutionof the adsorption resin will be concentrated to obtain pravastatinsodium concentrate. And water-soluble anti-solvent is added into theconcentrate for precipitation of pravastatin sodium. The addition of theanti-solvent will decrease the polarity of the concentrate and thepravastatin sodium is poorly soluble in such an anti-solvent, therebyaccelerating the precipitation of the pravastatin sodium in theconcentrate.

[0027] Or, an inorganic salt is added into the concentrate forsalting-out the precipitate of pravastatin sodium.

[0028] Finally, crystallization or re-crystallization is conducted toobtain the pravastatin sodium with high purity.

[0029] During the elution of the adsorption resin, the water-solubleorganic solvents may be selected from: methanol, acetone, or a mixtureof methanol and acetone.

[0030] The water-soluble anti-solvents added into the concentrate forprecipitation of pravartatin sodium may be selected from: ethanol,n-propanol, isopropanol, acetone and the mixture thereof. The acetone ismost preferable.

[0031] The inorganic salts for salting out the pravastatin sodiumprecipitate may be selected from: ammonium sulfate, ammonium chloride,sodium sulfate, sodium chloride and potassium chloride. The sodiumchloride is most preferable.

[0032] The present invention will be further described in detail in thefollowing examples, which are provided to exemplify the invention, butnot to limit the scope of the present invention.

EXAMPLE 1

[0033] A fermentation broth containing pravastatin sodium of 10 litersis centrifuged to remove microbial cell mass. The supernatant is addedwith 10 N sodium hydroxide solution to be basic of pH 12.0, which isthen pumped at constant rate and loaded to a resin column of Diaion®HP-20 (¢×H=5×60 cm; Bed vol=1.0 L; S.V.=3.0). The resin is then washedwith 7 liters of 0.1% (w/w) sodium hydroxide solution, and eluted withan eluant of 50% (w/w) methanol to obtain the pravastatin sodium elute.The elute is assayed by HPLC analysis to obtain crude pravastatin sodiumwith yield: 99.05% and purity: 94.0%.

[0034] The pravastatin sodium elute is adjusted to pH 9.0 by adding 3Nhydrochloric acid therein, and further concentrated to 30% (w/w)pravastatin sodium by a concentrator under reduced pressure. Theconcentrate is added therein with 4.0 liters acetone and stirred for 4hours at 25˜30° C. It is continuously stirred for next 2 hours atcooling temperature of 0˜5° C. to precipitate the pravastatin sodium.Vacuum filtration is done to obtain the filtered cake containingpravastatin sodium.

[0035] The filtered cake is slightly washed with little amount ofacetone and dissolved in 120 ml water, added with 1.0 g activated carbonand stirred for 2 hours for decoloring. The solution is then filteredand the filtrate is concentrated to 25% (w/w) pravastatin sodium, 4.0liters acetone is added into the concentrate and stirred under roomtemperature for 3 hours. Then, it is cooled to 0˜5° C. and stirred for 2hours to crystallize the pravastatin sodium, which is filtered undervacuum, dried to obtain purified pravastatin sodium: 18.3 g; overallyield: 61%; and purity: 98.5%.

EXAMPLE 2

[0036] A fermentation broth containing pravastatin sodium of 10 litersis centrifuged to remove microbial cell mass. The supernatant is addedwith 10 N sodium hydroxide solution to be basic of pH 11.0, which isthen pumped at constant rate and loaded to a resin column of Amberlite®AD-16 (¢×H=5×70 cm; Bed vol=1.2 L; S.V.=3.0). The resin is then washedwith 8 liters of 0.1% (w/w) sodium hydroxide solution, and eluted withan eluant of 50% (w/w) methanol to obtain the pravastatin sodium elute.

[0037] The pravastatin sodium elute is adjusted to pH 9.0 by adding 3Nhydrochloric acid therein, and further concentrated to 3.0% (w/w)pravastatin sodium. The concentrate is added therein with 300 g sodiumchloride and stirred for 4 hours at 25˜30° C. It is continuously stirredfor next 2 hours at cooling temperature of 0˜5° C. to salt out thepravastatin sodium precipitate. Filtration is done to obtain thefiltered cake containing pravastatin sodium, purity: 99.3%.

[0038] The salting-out cake is dissolved in 120 ml water, added with 1.0g activated carbon and stirred for 2 hours for decoloring. The solutionis then filtered and the filtrate is then desalted by XAD-4 or HP-20ss.The pravastatin sodium elute is concentrated under reduced pressure to30% (w/w) and 4.0 liters acetone is added into the concentrate andstirred at 25˜30° C. for 4 hours. Then, it is cooled to 0˜5° C. andstirred for 2 hours to crystallize the pravastatin sodium, which isfiltered under vacuum, dried to obtain purified pravastatin sodium: 16.5g; overall yield: 55%; and purity: 99.5%.

EXAMPLE 3

[0039] A fermentation broth containing pravastatin sodium of 10 litersis centrifuged to remove microbial cell mass. The supernatant is addedwith 10 N sodium hydroxide solution to be basic of pH 11.0, which isthen pumped at constant rate and loaded to a resin column of Diaion®SP-200 (¢×H=5×60 cm; Bed vol=1.0 L; S.V.=3.0). The resin is then washedwith 7 liters of 0.1% (w/w) sodium hydroxide solution, and eluted withan eluant of 50% (w/w) methanol to obtain the pravastatin sodium elute.The elute is assayed by HPLC analysis to obtain crude pravastatin sodiumwith yield: 99% and purity: 95%.

[0040] The pravastatin sodium elute is adjusted to pH 9.0 by adding 3Nhydrochloric acid therein, and further concentrated to 3.0% (w/w)pravastatin sodium by a concentrator under reduced pressure. Theconcentrate is added therein 280 g sodium chloride and stirred for 4hours at 25˜30° C. It is continuously stirred for next 2 hours atcooling temperature of 0˜5° C. to precipitate the pravastatin sodium.Filtration is done to obtain the filtered cake containing pravastatinsodium.

[0041] The filtered cake is dissolved in 120 ml water, added with 1.2 gactivated carbon and stirred for 2 hours for decoloring. The solution isthen filtered and desalted by HP-20ss for de-salting. The pravastatinsodium elute is concentrated under reduced pressure to 30% (w/w)pravastatin sodium. 4.0 liters acetone is added into the concentrate andstirred at 25˜30° C. for 4 hours. Then, it is cooled to 0˜5° C. andstirred for 2 hours to crystallize the pravastatin sodium, which isfiltered, dried to obtain purified pravastatin sodium: 17.0 g; overallyield: 56.7%; and purity: 99.6%.

EXAMPLE 4

[0042] A fermentation broth containing pravastatin sodium of 1000 litersis centrifuged to remove microbial cell mass. The supernatant is addedwith 45% (w/w) sodium hydroxide solution to be basic of pH 12.0, whichis then pumped at constant rate and loaded to a resin column of Diaion®HP-20 (Bed vol=100 L; S.V.=3.0). The resin is then washed with 800liters of 0.1% (w/w) sodium hydroxide solution, and eluted with aneluant of 50% (w/w) methanol to obtain the pravastatin sodium elute. Theelute is assayed by HPLC analysis to obtain crude pravastatin sodiumwith yield: 98.5% and purity: 94.5%.

[0043] The pravastatin sodium elute is adjusted to pH 9.0 by adding 6Nhydrochloric acid therein, and further concentrated to 3.0% (w/w)pravastatin sodium. The concentrate is added therein with 30 kg sodiumchloride and stirred for 4˜6 hours at 25˜30° C. It is continuouslystirred for next 3 hours at cooling temperature of 0˜5° C. toprecipitate the pravastatin sodium. Centrifugation is done to obtain thefiltered cake containing pravastatin sodium, purity: 99.4% by HPLC.

[0044] The filtered cake may be decolored, if necessarily. The cake isdissolved in 20 liters water and desalted by HP-20ss for de-salting. Thepravastatin sodium elute is concentrated under reduced pressure to 30%(w/w) pravastatin sodium. 400 liters acetone is added into theconcentrate and stirred at 25˜30° C. for 4-6 hours. Then, it is cooledto 0˜5° C. and stirred for 3 hours to crystallize the pravastatinsodium, which is filtered, dried to obtain purified pravastatin sodium:1.68 kg; overall yield: 56%; and purity: 99.57%.

[0045] The present invention may produce pravastatin sodium with highpurity and high yield. Unexpected impurities can be prevented in thepurification process. Meanwhile, the solvents as used in the process aresafe, not hazardous to the environment and the solvent consumption iscontrolled at a very reasonable rate. So, the present invention may becommercialized economically, without conflicting the environmentalprotection.

[0046] The present invention may be modified without departing from thespirit and scope of the present invention.

I claim:
 1. A process for stably purifying pravastatin sodium comprisingthe steps of: a. clarifying a fermentation broth containing pravastatinsodium by a step selected from centrifugation and filtration to obtain aclear solution containing the pravastatin sodium; and adjusting theclear solution to be basic having pH value ranging from pH 10˜13; b.adsorbing the pravastatin sodium with non-ionic resin which is thenwashed with water; eluting the pravastatin sodium as adsorbed on theresin by water-soluble organic solvent to obtain pravastatin sodiumfraction; and concentrating the fraction to be pravastatin sodiumconcentrate; c. treating the concentrate to form a precipitate ofpravastatin sodium; and d. recrystallizing the precipitate for makingpravastatin sodium crystal with high purity and high yield.
 2. A processaccording to claim 1, wherein the water-soluble organic solvent, as usedin the step (b) for eluting the pravastatin sodium, is selected from:methanol, acetone and a mixture of methanol and acetone.
 3. A processaccording to claim 1, wherein the treating of said concentrate forforming a precipitate of pravastatin sodium in step (c) comprising theaddition of water-soluble anti-solvent into said concentrate.
 4. Aprocess according to claim 3, wherein said water-soluble anti-solvent isselected from: ethanol, n-propanol, isopropanol, acetone and the mixturethereof.
 5. A process according to claim 1, wherein the treating of saidconcentrate for forming a precipitate of pravastatin sodium in step (c)comprising the addition of inorganic salt for salting out thepravastatin sodium precipitate.
 6. A process according to claim 5,wherein said inorganic salt is selected form: ammonium sulfate, ammoniumchloride, sodium sulfate, sodium chloride and potassium chloride.